As one of methods for measuring a chemical substance in a liquid sample, methods using a molecule-recognizing material are known. As one of the methods using the molecule-recognizing material, an immunosensor utilizing an antigen-antibody reaction is used.
Since an antibody has a strong molecule recognizing characteristic, and techniques for preparing an antibody specifically binding to a component to be measured have been developed, an immunosensor using an antibody has been increasingly used for detection of a virus or for measurement or rapid diagnosis of a disease marker or an environmental pollutant.
For example, in a sandwich-type enzyme-linked immunosorbent assay (ELISA) commonly used as an immunoassay, a plurality of kinds of antibodies that bind to different sites in one antigen need to be prepared, and a labeling molecule needs to be bound to one of these antibodies.
Furthermore, in this sandwich-type ELISA, 1) antibodies specifically binding to a target antigen are prepared on a solid phase; 2) a solution containing a target substance is added to this solid phase, and the antigen is allowed to bind to the solid phase by an antigen-antibody reaction; 3) excess proteins and antigens that have not bound to the solid phase are removed by washing; 4) then a labeled antibody is added to solid phase and allowed to bind to the antigen; 5) and the antigen is quantified using the immobilized labeled antibody.
Therefore, to perform an analysis with this operating procedure, pipetting, washing, and exchanging a liquid are required, and an absorption spectrometer or a fluorescence spectrometer is required for the final measurement. Therefore, ELISA suffers from problems such as a high testing cost and a complex procedure.
Meanwhile, in the field of immunoassay, an immunological chromatographic method has been developed as a simple and inexpensive method (for example, refer to Patent Document 1).
The immunological chromatographic method is a measurement method for detecting the presence of an antigen with the following mechanism of action. 1) A sample such as urine, blood, or a mucous membrane suspension containing an antigen to be measured is added dropwise onto a sample drop section of a filter paper containing a gold colloid-labeled antibody at a predetermined position and developed. 2) When the sample containing the antigen is developed, the antigen is brought into contact with the gold colloid-labeled antibody, and the gold colloid-labeled antibody binds to the antigen by an antigen-antibody reaction. 3) When the gold colloid-labeled antibody bound to the antigen is developed, the antibody binds to a second antibody immobilized beforehand at a predetermined position of the filter paper, and gold colloid is accumulated in this portion. 4) Accumulation of gold colloid results in purple-red coloration. The presence of the antigen is detected by accumulation of gold colloid.
Since chromatography using a filter paper is utilized in this method, there is a separation action of separating the measurement object and impurities, and even a sample containing many impurities can be measured. Furthermore, since no special device is required, and a result can be visually determined, the method is used in testing for influenza virus, pregnancy test, and the like as a simple method for measuring an antigen.
Furthermore, in the cases where measurement of a trace amount is required, for example, introduction of a label into an antibody causes a loss of the antibody activity or a change in recognition of an antigen, a small amount of the antibody is obtained, or the like, a special measurement method is required.
As one of such measurement methods, a measurement chip is used in which a filter paper is placed in a plastic passage (for example, refer to Non-patent Document 1). In this measurement chip, a component to be measured is separated from impurities by development in the planar surface of the filter paper and then led to a clear chamber, and the component to be measured is quantified by intensity of a light transmitted through the chamber.
One of methods of measuring a biomolecular interaction such as an antigen-antibody reaction using an unlabeled antibody or antigen or DNA hybridization is an SPR measurement method utilizing effects of a surface plasmon resonance, and an SPR sensor is used in this measurement method.
The SPR sensor is an immunosensor utilizing an antigen-antibody reaction, which comprises a metal thin film on which an antigen or an antibody is immobilized. The antigen or the antibody immobilized on the metal thin film binds to an antibody or an antigen by an antigen-antibody reaction. When the antigen or the antibody binds to an antibody or an antigen by an antigen-antibody reaction, a change occurs in a refractive index. The antibody or the antigen is detected by detecting the change in the refractive index. Since it is unnecessary to label a molecule to be detected with a radioactive substance or a fluorescent substance, and high concentration sensitivity can be obtained, the SPR sensor has been increasingly used as means for detecting a biologically-relevant substance (for example, refer to Patent Document 2).
The SPR measurement is characterized in that only a small volume of a sample required is necessary for measurement since a refractive index is measured in a range of a volume that an evanescent light reaches in a portion on the gold thin film on which a light is reflected. Furthermore, in the SPR measurement, an optical system for measuring an SPR phenomenon and a measurement element for performing an antigen-antibody reaction can be separated using a transparent substrate, and replacement of the measurement element enables measurement of a different molecule sample as well as prevention of contamination of the measurement device. In particular, when a sample contains a toxin, leakage of the toxin can be prevented. Furthermore, a plurality of samples can be measured at the same time in the SPR measurement, resulting in a high measurement throughput.    Patent Document 1: Japanese Patent Publication No. H07-36017 (1995)    Patent Document 2: Japanese Patent No. 3294605    Non-patent Document 1: Anal. Chem. 2005, 77, 7901-7907